Fig. 7: RNF181 facilitates K63-linked poly-ubiquitination of ERα.

a RNF181 decreases poly-ubiquitination of ERα. HEK293 cells were transfected with 2 µg ERα plasmid, 0.5 µg HA Ub plasmid and 0.5 µg Myc-tag or Myc-RNF181 plasmids. The cell extracts were immunoprecipitated with HA antibody. The poly-ubiquitinated ERα was detected via western blotting analysis. b RNF181 decreases K48-linked poly-ubiquitination of ERα. HEK293 cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-K48 Ubi plasmid and 0.5 µg Myc-tag or Myc-RNF181 plasmids. The cell extracts were immunoprecipitated with HA antibody. The K48 specific poly-ubiquitinated ERα was detected via western blotting analysis. c RNF181 promotes K63-linked poly-ubiquitination of ERα. HEK293 cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-K63 Ubi plasmid and 0.5 µg Myc-tag or Myc-RNF181 plasmids. The cell extracts were immunoprecipitated with HA antibody. The K63 specific poly-ubiquitinated ERα was detected via western blotting analysis. d RNF181 depletion increases endogenous K48-linked poly-ubiquitination of ERα in breast cancer cells. MCF-7 cells were transfected with 50 uM siRNF181 siRNA or siControl. After 48 h, cells were treated with MG132 for 6 h. The cell extracts were immunoprecipitated with ERα antibody (D8H8, Cell signaling Technology). The K48 specific poly-ubiquitinated ERα was detected via K48 specific Ub antibody (A101, R&D company). e RNF181 depletion inhibited endogenous K63-linked poly-ubiquitination of ERα in breast cancer cells. MCF-7 cells were transfected with 50 uM siRNF181 siRNA or siControl. After 48 h, cells were treated with MG132 for 6 h. The cell extracts were immunoprecipitated with ERα antibody (D8H8, Cell signaling Technology). The K63 specific poly-ubiquitinated ERα was detected via K63 specific Ub antibody (HWA404, Enzo life science). f The intact RNF181 protein is require for the inhibition of K48-linked poly-ubiquitination on ERα. HEK293 cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-K48 Ubi plasmid and 0.5 µg GFP-RNF181 full length or mutants (ΔN-terminal, ΔRING + ΔN-terminal, ΔC-terminal, ΔRING + ΔC-terminal) plasmids. The cell extracts were immunoprecipitated with HA antibody. The K48 specific poly-ubiquitinated ERα was detected via western blotting analysis. g The intact RNF181 protein is require for the induction of K63-linked poly-ubiquitination on ERα. HEK293 cells were transfected with 2 µg ERα plasmid, 0.5 µg HA-K63 Ubi plasmid and 0.5 µg GFP-RNF181 full length or mutants (ΔN-terminal, ΔRING + ΔN-terminal, ΔC-terminal, ΔRING + ΔC-terminal) plasmids. The cell extracts were immunoprecipitated with HA antibody. The K63 specific poly-ubiquitinated ERα was detected via western blotting analysis.